One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as protein phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of MAP kinases have been identified and each of these consists of a specific module of kinases that function downstream of an activating stimulus. One subgroup of the MAP kinases is the p38 MAP kinase cascade which has been shown to be activated by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 as well as environmental stress (e.g., osmotic shock and ultraviolet radiation). Upon activation, the p38 cascade leads to the induction of gene expression of several factors involved in inflammation and immunity including TNF, interleukin-6, granulocyte-macrophage colony stimulating factor (GM-CSF), and HIV long terminal repeat (Paul et al., Cell Signal., 1997, 9, 403-410). Modulation of the expression of one or more of the kinases in the p38 pathway is desireable in order to interfere with inflammatory or apoptotic responses associated with disease states. Various inhibitors have been shown to have efficacy in animal models of arthritis (Badger et al., J. Pharmacol. Exp. Ther., 1996, 279, 1453-1461) and angiogenesis (Jackson et al., J. Pharmacol. Exp. Ther., 1998, 284, 687-692). MacKay, K. and Mochy-Rosen, D. demonstrate that an inhibitor of p38 MAP kinases prevents apoptosis during ischemia in cardiac myocytes, suggesting that p38 MAP kinase inhibitors can be used for treating ischemic heart disease (Mackay and Mochly-Rosen, J. Biol. Chem., 1999, 274, 6272-6279). p38 MAP kinase is also required for T-cell HIV-1 replication (Cohen et al., Molecular Medicine, 1997, 3, 339-346) and may be a useful target for AIDS therapy. Disclosed in PCT application WO 98/54203 are methods to increase cancer cell sensitivity to cancer therapy by contacting said cells with a p38 pathway inhibitor. These inhibitors being ribozymes, antisense nucleic acid molecules targeting MEKK1, or dominant negative mutants of MEKK1 (Mercola, 1998). However, within this PCT publication, the composition of these inhibitors is not disclosed.
MAP kinase kinase 6 (also known as MKK6, MEK6 and SAPKK3) is a kinase within the p38 cascade that specifically activates all three isoforms of p38 MAP kinase (Cuenda et al., Embo J., 1997, 16, 295-305; Enslen et al., J. Biol. Chem., 1998, 273, 1741-1748; Goedert et al., Embo J., 1997, 16, 3563-3571; Raingeaud et al., Mol. Cell. Biol., 1996, 16, 1247-1255). MAP kinase kinase 6 was originally cloned from skeletal muscle using a probe to MKK3, a MAP kinase that also activates p38 and is very similar to MAP kinase kinase 6 (Raingeaud et al., Mol. Cell. Biol., 1996, 16, 1247-1255). Characterization of the MAP kinase kinase 6 protein and nucleotide sequence revealed that MAP kinase kinase 6 exists in a variety of alternatively spliced forms with distinct patterns of expression (Han et al., J. Biol. Chem., 1996, 271, 2886-2891). One variant, with an mRNA of 1.7 kilobases was found exclusively in skeletal muscle while other variants were found in the heart liver and kidney. This differential expression pattern implies that MAP kinase kinase 6 may play unique roles in these tissues (Han et al., J. Biol. Chem., 1996, 271, 2886-2891).
Disclosed in the PCT publication WO 97/22704 are the nucleic acid sequence encoding MAP kinase kinase 6, compositions and methods of modulating the p38 MAP kinase pathway, including antibodies to MAP kinase kinase 6, and a kit to identify agents that modulate the pathway (Stein and Yang, 1997).
Disclosed in U.S. Pat. No. 5,736,381 are MKK isoforms including MAP kinase kinase 6, a polynucleotide encoding MAP kinase kinase 6, a vector encoding MAP kinase kinase 6 and host cells expressing said vector (Davis et al., 1998). Also disclosed are polynucleotides fully complementary to MAP kinase kinase 6 or variants thereof (Davis et al., 1996). Antisense oligonucleotides to MAP kinase kinase 6 are generally disclosed. Further disclosed in the PCT publication WO 96/36642 are antibodies to MAP kinase kinase 6 and methods to measure MAP kinase kinase 6 activity and synthesis (Davis et al., 1996).
The effect of TNF on MAP kinase kinase 6 activation and signaling has been widely investigated. Subtype specific cytokine activation of a MAP kinase pathway has been described in neutrophils by Suzuki et al. who demonstrated that the MAP kinase kinase 6 is specifically activated by the TNF-.alpha. and granulocyte-macrophage colony stimulating factor (GM-CSF) in the p38 pathway (Suzuki et al., Blood, 1999, 93, 341-349). Studies of dominant negative and dominant active mutants of MAP kinase kinase 6 in endothelial cells showed that activation of the MKK6/p38 pathway is critical for the TNF-.alpha. mediated expression of monocyte chemoattractant protein 1 (MCP-1) (Goebeler et al., Blood, 1999, 93, 857-865). MCP-1 recruits leukocytes to the site of inflammation and excesses of this protein have been detected in diseases such as atherosclerosis.
The MAP kinase kinase 6 protein has also been shown to regulate signaling events associated with apoptosis. Zechner et al. demonstrated that MAP kinase kinase 6 activates NF-kappa-B and inhibits apoptosis in rat myocardial cells (Zechner et al., J. Biol. Chem., 1998, 273, 8232-8239). Myocardial cell apoptosis is believed to contribute to several cardiac disorders including heart failure and myocardial infarction. In these studies, overexpression of a constitutively active mutant of MAP kinase kinase 6 protected cells from both anisomycin- and MEKK1-induced apoptosis (Zechner et al., J. Biol. Chem., 1998, 273, 8232-8239).
Mutant forms of MAP kinase kinase 6 have also been used to demonstrate the role of MAP kinase kinase 6 in hematopoiesis. Forced activation of the p38 cascade using a gain-of-function MAP kinase kinase 6 mutant was shown to induce erythroid differentiation in SKT6 cells (Nagata et al., Blood, 1998, 92, 1859-1869).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MAP kinase kinase 6 and to date, strategies aimed at modulating MAP kinase kinase 6 function have involved the use of antibodies, dominant negative and dominant active mutants of the protein and MAP kinase pathway inhibitors. There remains, consequently, a long felt need for additional agents capable of effectively inhibiting MAP kinase kinase 6 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of MAP kinase kinase 6 expression.
The present invention provides compositions and methods for modulating MAP kinase kinase 6 expression, including modulation of the alternatively spliced form of MAP kinase kinase 6, MKK6b.